Chuanlun L. Zhang

Global Occurrence of Archaeal amoA Genes in Terrestrial Hot Springs

ABSTRACT Despite the ubiquity of ammonium in geothermal environments and the thermodynamic favorability of aerobic ammonia oxidation, thermophilic ammonia-oxidizing microorganisms belonging to the crenarchaeota kingdom have only recently been described. In this study, we analyzed microbial mats and surface sediments from 21 hot spring samples (pH 3.4 to 9.0; temperature, 41 to 86°C) from the United States, China, and Russia and obtained 846 putative archaeal ammonia monooxygenase large-subunit (amoA) gene and transcript sequences, representing a total of 41 amoA operational taxonomic units (OTUs) at 2% identity. The amoA gene sequences were highly diverse, yet they clustered within two major clades of archaeal amoA sequences known from water columns, sediments, and soils: clusters A and B. Eighty-four percent (711/846) of the sequences belonged to cluster A, which is typically found in water columns and sediments, whereas 16% (135/846) belonged to cluster B, which is typically found in soils and sediments. Although a few amoA OTUs were present in several geothermal regions, most were specific to a single region. In addition, cluster A amoA genes formed geographic groups, while cluster B sequences did not group geographically. With the exception of only one hot spring, principal-component analysis and UPGMA (unweighted-pair group method using average linkages) based on the UniFrac metric derived from cluster A grouped the springs by location, regardless of temperature or bulk water pH, suggesting that geography may play a role in structuring communities of putative ammonia-oxidizing archaea (AOA). The amoA genes were distinct from those of low-temperature environments; in particular, pair-wise comparisons between hot spring amoA genes and those from sympatric soils showed less than 85% sequence identity, underscoring the distinctness of hot spring archaeal communities from those of the surrounding soil system. Reverse transcription-PCR showed that amoA genes were transcribed in situ in one spring and the transcripts were closely related to the amoA genes amplified from the same spring. Our study demonstrates the global occurrence of putative archaeal amoA genes in a wide variety of terrestrial hot springs and suggests that geography may play an important role in selecting different assemblages of AOA.

Nonmarine Crenarchaeol in Nevada Hot Springs

ABSTRACT Glycerol dialkyl glycerol tetraethers (GDGTs) are core membrane lipids of the Crenarchaeota. The structurally unusual GDGT crenarchaeol has been proposed as a taxonomically specific biomarker for the marine planktonic group I archaea. It is found ubiquitously in the marine water column and in sediments. In this work, samples of microbial community biomass were obtained from several alkaline and neutral-pH hot springs in Nevada, United States. Lipid extracts of these samples were analyzed by high-performance liquid chromatography-mass spectrometry and by gas chromatography-mass spectrometry. Each sample contained GDGTs, and among these compounds was crenarchaeol. The distribution of archaeal lipids in Nevada hot springs did not appear to correlate with temperature, as has been observed in the marine environment. Instead, a significant correlation with the concentration of bicarbonate was observed. Archaeal DNA was analyzed by denaturing gradient gel electrophoresis. All samples contained 16S rRNA gene sequences which were more strongly related to thermophilic crenarchaeota than to Cenarchaeum symbiosum, a marine nonthermophilic crenarchaeon. The occurrence of crenarchaeol in environments containing sequences affiliated with thermophilic crenarchaeota suggests a wide phenotypic distribution of this compound. The results also indicate that crenarchaeol can no longer be considered an exclusive biomarker for marine species.

Alkaline anaerobic respiration : isolation and characterization of a novel alkaliphilic and metal-reducing bacterium

ABSTRACT Iron-reducing enrichments were obtained from leachate ponds at the U.S. Borax Company in Boron, Calif. Based on partial small-subunit (SSU) rRNA gene sequences (approximately 500 nucleotides), six isolates shared 98.9% nucleotide identity. As a representative, the isolate QYMF was selected for further analysis. QYMF could be grown with Fe(III)-citrate, Fe(III)-EDTA, Co(III)-EDTA, or Cr(VI) as electron acceptors, and yeast extract and lactate could serve as electron donors. Growth during iron reduction occurred over the pH range of 7.5 to 11.0 (optimum, pH 9.5), a sodium chloride range of 0 to 80 g/liter (optimum, 20 g/liter), and a temperature range of 4 to 45°C (optimum, approximately 35°C), and iron precipitates were formed. QYMF was a strict anaerobe that could be grown in the presence of borax, and the cells were straight rods that produced endospores. Sodium chloride and yeast extract stimulated growth. Phylogenetic analysis of the SSU rRNA gene indicated that the bacterium was a low-G+C gram-positive microorganism and had 96 and 92% nucleotide identity with Alkaliphilus transvaalensis and Alkaliphilus crotonatoxidans, respectively. The major phospholipid fatty acids were 14:1, 16:1ω7c, and 16:0, which were different from those of other alkaliphiles but similar to those of reported iron-reducing bacteria. The results demonstrated that the isolate might represent a novel metal-reducing alkaliphilic species. The name Alkaliphilus metalliredigens sp. nov. is proposed. The isolation and activity of metal-reducing bacteria from borax-contaminated leachate ponds suggest that bioremediation of metal-contaminated alkaline environments may be feasible and have implications for alkaline anaerobic respiration.

Factors Controlling the Distribution of Archaeal Tetraethers in Terrestrial Hot Springs

ABSTRACT Glycerol dialkyl glycerol tetraethers (GDGTs) found in hot springs reflect the abundance and community structure of Archaea in these extreme environments. The relationships between GDGTs, archaeal communities, and physical or geochemical variables are underexamined to date and when reported often result in conflicting interpretations. Here, we examined profiles of GDGTs from pure cultures of Crenarchaeota and from terrestrial geothermal springs representing a wide distribution of locations, including Yellowstone National Park (United States), the Great Basin of Nevada and California (United States), Kamchatka (Russia), Tengchong thermal field (China), and Thailand. These samples had temperatures of 36.5 to 87°C and pH values of 3.0 to 9.2. GDGT abundances also were determined for three soil samples adjacent to some of the hot springs. Principal component analysis identified four factors that accounted for most of the variance among nine individual GDGTs, temperature, and pH. Significant correlations were observed between pH and the GDGTs crenarchaeol and GDGT-4 (four cyclopentane rings, m/z 1,294); pH correlated positively with crenarchaeol and inversely with GDGT-4. Weaker correlations were observed between temperature and the four factors. Three of the four GDGTs used in the marine TEX86 paleotemperature index (GDGT-1 to -3, but not crenarchaeol isomer) were associated with a single factor. No correlation was observed for GDGT-0 (acyclic caldarchaeol): it is effectively its own variable. The biosynthetic mechanisms and exact archaeal community structures leading to these relationships remain unknown. However, the data in general show promise for the continued development of GDGT lipid-based physiochemical proxies for archaeal evolution and for paleo-ecology or paleoclimate studies.

Lipid and carbon isotopic evidence of methane-oxidizing and sulfate-reducing bacteria in association with gas hydrates from the Gulf of Mexico

An integrated lipid biomarker–carbon isotope approach reveals new insight to microbial methane oxidation in the Gulf of Mexico gas-hydrate system. Hydrate-bearing and hydrate-free sediments were collected from the Gulf of Mexico slope using a research submersible. Phospholipid fatty acids consist mainly of C16–C18 compounds, which are largely derived from bacteria. The phospholipid fatty acids suggest that total biomass is enhanced 11–30-fold in gas-hydrate–bearing sediment compared to hydrate-free sediment. Lipid biomarkers indicative of sulfate-reducing bacteria are strongly depleted in 13C (δ13C = −48‰ to −70‰) in the hydrate-bearing samples, suggesting that they are involved in the oxidation of methane (δ13C = −47‰ for thermogenic methane and −70‰ for biogenic methane). Isotopic properties of other biomarkers suggest that sulfur-oxidizing bacteria ( Beggiatoa ) may also contribute to the lipid pool in hydrate-bearing samples, which are characterized by less negative δ13C values (to −11.2‰). In the hydrate-free sample, fatty acid biomarkers have δ13C values of −27.6‰ to −39.6‰, indicating that crude oil (average ∼−27‰) or terrestrial organic carbon (average ∼−20‰) are the likely carbon sources. Our results provide the first lipid biomarker–stable isotope evidence that sulfate- reducing bacteria play an important role in anaerobic methane oxidation in the Gulf of Mexico gas hydrates. The coupled activities of methane-oxidizing and sulfate-reducing organisms contribute to the development of ecosystems in deep-sea environments and result in sequestration of carbon as buried organic carbon and authigenic carbonates. These have implications for studying climate change based on carbon budgets.

Thermophilic Temperature Optimum for Crenarchaeol Synthesis and Its Implication for Archaeal Evolution

ABSTRACT The isoprenoid lipid crenarchaeol is widespread in hot springs of California and Nevada. Terrestrial and marine data together suggest a maximum relative abundance of crenarchaeol at ∼40°C. This warm temperature optimum may have facilitated colonization of the ocean by (hyper)thermophilic Archaea and the major marine radiation of Crenarchaeota.

Isolation, Characterization, and Ecology of Sulfur-Respiring Crenarchaea Inhabiting Acid-Sulfate-Chloride-Containing Geothermal Springs in Yellowstone National Park

ABSTRACT Elemental sulfur (S0) is associated with many geochemically diverse hot springs, yet little is known about the phylogeny, physiology, and ecology of the organisms involved in its cycling. Here we report the isolation, characterization, and ecology of two novel, S0-reducing Crenarchaea from an acid geothermal spring referred to as Dragon Spring. Isolate 18U65 grows optimally at 70 to 72°C and at pH 2.5 to 3.0, while isolate 18D70 grows optimally at 81°C and pH 3.0. Both isolates are chemoorganotrophs, dependent on complex peptide-containing carbon sources, S0, and anaerobic conditions for respiration-dependent growth. Glycerol dialkyl glycerol tetraethers (GDGTs) containing four to six cyclopentyl rings were present in the lipid fraction of isolates 18U65 and 18D70. Physiological characterization suggests that the isolates are adapted to the physicochemical conditions of Dragon Spring and can utilize the natural organic matter in the spring as a carbon and energy source. Quantitative PCR analysis of 16S rRNA genes associated with the S0 flocs recovered from several acid geothermal springs using isolate-specific primers indicates that these two populations together represent 17 to 37% of the floc-associated DNA. The physiological characteristics of isolates 18U65 and 18D70 are consistent with their potential widespread distribution and putative role in the cycling of sulfur in acid geothermal springs throughout the Yellowstone National Park geothermal complex. Based on phenotypic and genetic characterization, the designations Caldisphaera draconis sp. nov. and Acidilobus sulfurireducens sp. nov. are proposed for isolates 18U65 and 18D70, respectively.

Lipid Biomarkers and Carbon Isotope Signatures of a Microbial (Beggiatoa) Mat Associated with Gas Hydrates in the Gulf of Mexico

ABSTRACT White and orange mats are ubiquitous on surface sediments associated with gas hydrates and cold seeps in the Gulf of Mexico. The goal of this study was to determine the predominant pathways for carbon cycling within an orange mat in Green Canyon (GC) block GC 234 in the Gulf of Mexico. Our approach incorporated laser-scanning confocal microscopy, lipid biomarkers, stable carbon isotopes, and 16S rRNA gene sequencing. Confocal microscopy showed the predominance of filamentous microorganisms (4 to 5 μm in diameter) in the mat sample, which are characteristic of Beggiatoa. The phospholipid fatty acids extracted from the mat sample were dominated by 16:1ω7c/t (67%), 18:1ω7c (17%), and 16:0 (8%), which are consistent with lipid profiles of known sulfur-oxidizing bacteria, including Beggiatoa. These results are supported by the 16S rRNA gene analysis of the mat material, which yielded sequences that are all related to the vacuolated sulfur-oxidizing bacteria, including Beggiatoa, Thioploca, and Thiomargarita. The δ13C value of total biomass was −28.6‰; those of individual fatty acids were −29.4 to −33.7‰. These values suggested heterotrophic growth of Beggiatoa on organic substrates that may have δ13C values characteristic of crude oil or on their by-products from microbial degradation. This study demonstrated that integrating lipid biomarkers, stable isotopes, and molecular DNA could enhance our understanding of the metabolic functions of Beggiatoa mats in sulfide-rich marine sediments associated with gas hydrates in the Gulf of Mexico and other locations.

Archaeal lipid biomarkers and isotopic evidence of anaerobic methane oxidation associated with gas hydrates in the Gulf of Mexico

Abstract Anaerobic oxidation of methane (AOM) occurs in the Gulf of Mexico gas hydrate systems. Here we show lipid biomarker and isotopic evidence that archaea are involved in AOM. The estimated abundance of total archaeal lipids ranges from 44.8 to 60.4 μg/g (dry sediment) in hydrate-bearing samples but is below detection limit in the hydrate-free sample. The δ13C values of archaeal lipids range from −69 to −99 ‰ in hydrate-bearing samples. These results suggest that biomass of archaea is significantly enhanced through AOM at the gas hydrate deposits. These data also support a currently acknowledged mechanism of AOM mediated by a consortium of sulfate-reducing bacteria and archaea observed in a variety of methane-rich marine settings. Anaerobic oxidation of oil hydrocarbons also occurs in the Gulf of Mexico gas hydrate systems as shown by degradation of n-alkanes (>C15) in the anoxic sediments. These processes convert hydrocarbons to carbon dioxide and increase pore water alkalinity, which promote the precipitation of enormous volumes of authigenic carbonate rock depleted in 13C. This long-term geologic sequestration of carbon may affect models of global climate change.

Temperature-dependent oxygen and carbon isotope fractionations of biogenic siderite

Isotopic compositions of biogenic iron minerals may be used to infer environmental conditions under which bacterial iron reduction occurs. The major goal of this study is to examine temperature-dependent isotope fractionations associated with biogenic siderite (FeCO 3). Experiments were performed by using both mesophilic (,35°C) and thermophilic (.45°C) iron-reducing bacteria. In addition, control experiments were performed to examine fractionations under nonbiologic conditions. Temperature-dependent oxygen isotope fractionation occurred between biogenic siderite and water from which the mineral was precipitated. Samples in thermophilic cultures (45-75°C) gave the best linear correlation, which can be described as 10 3 lnasid-wt 5 2.56 3 10 6 T 22 (K) 1 1.69. This empirical equation agrees with that derived from inorganically precipitated siderite by Carothers et al. (1988) and may be used to approximate equilibrium fractionation. Carbon isotope fractionation between biogenic siderite and CO 2, based on limited data, also varied with temperature and was consistent with the inorganically precipitated siderite of Carothers et al. (1988). These results indicate that temperature is a controlling factor for isotopic variations in biogenic minerals examined in this study. The temperature-dependent fractionations under laboratory conditions, however, could be complicated by other factors including incubation time and concentration of bicarbonate. Early precipitated siderite at 120-mM initial bicarbonate tended to be enriched in 18 O. Siderite formed at ,30 mM of bicarbonate tended to be depleted in 18 O. Other variables, such as isotopic compositions of water, types of bacterial species, or bacterial growth rates, had little effect on the fractionation. In addition, siderite formed in abiotic controls had similar oxygen isotopic compositions as those of biogenic siderite at the same temperature, suggesting that microbial fractionations cannot be distinguished from abiotic fractionations under conditions examined here. Copyright